RNA interference (RNAi) and related pathways trigger post-transcriptional gene silencing using single-stranded guide RNAs that base pair with cognate mRNAs to direct their endonucleolytic cleavage or translational repression by RNA-induced silencing complexes (RISCs). Silencing is initiated by long dsRNAs or RNA hairpins, which are processed by the endonuclease Dicer to yield 21-23 nt short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively. These small interfering dsRNAs are then loaded onto Argonaute2 (Ago2), the endonuclease component of RISC.
The eukaryotic endoribonuclease Dicer recognizes distinct types of double-stranded RNA (dsRNA) substrates and generates ˜21 base pair products that assemble into RISCs. In humans, Dicer plays a central role in producing most of the small regulatory RNAs that enter this pathway in the cytoplasm. Structural analysis of Giardia Dicer and biochemical studies of human Dicer (hDicer) suggest that the enzyme functions as a monomer to bind, orient and cleave dsRNA substrates using a two-metal-ion mechanism similar to that of bacterial Ribonuclease III.
Although mammalian Dicer has been successfully produced recombinantly in eukaryotic cells, recombinant production of mammalian Dicer in prokaryotic cells has proved challenging.